Stem cells refer to cells having not only self-replicating ability but also the ability to differentiate into at least two types of cells, and can be divided into totipotent stem cells, pluripotent stem cells, and multipotent stem cells. In recent years, studies have been actively conducted to treat various diseases with stem cells capable of differentiating into various cells. Thus, the ultimate object of stem cell studies is to make a desired type of cell or tissue for use in technology such as cell therapy or tissue engineering.
Thus, the problem to be solved to use stem cells in actual applications is the development of a technology capable of inducing the differentiation of stem cells into desired cells. Accordingly, studies have been attempted to induce stem cells to differentiate into specific cells, and induced pluripotent stem (iPS) cells produced by reprogramming of somatic cells, etc., have been used in cell differentiation.
Generally, methods of promotes differentiation are used to induce the differentiation of stem cells. Typical examples of these methods include a method of inducing the differentiation of embryonic stem cells into neurons by using retionic acid (Dev. Dyn. 236:3255-3266, 2007), a method of inducing the differentiation of embryonic stem cells into hepatocytes by using activin A (Nat. Biotechnol. 23:1534-1541, 2005), a method of inducing the differentiation of embryonic stem cells into cardiomyocytes by using ascorbic acid (Circulation 107:1912-1916, 2003), and the like. However, conventional methods have disadvantages in that these methods are expensive due to the use of expensive reagents such as cytokines and show low differentiation rates. Thus, in order to effectively use stem cells in various fields, it is required to develop an inexpensive and easy method capable of inducing the differentiation of stem cells into a desired specific tissue with high differentiation efficiency.
In most vertebrates including humans, three-germ-layer cells (endoderm, mesoderm and ectoderm) are formed through gastrulation of early embryos, and all cells constituting the human body tissue differentiate from the three-germ-layer cells. In the case of embryonic stem cells established in vitro by extracting an inner cell mass from blastocysts that are early embryos, an event similar to gastrulation is observed during the formation of embryoid bodies. The first important step for inducing the differentiation of specific cells from embryonic stem cells is to increase a specific germ layer which is the origin of the target cells to be obtained by differentiation, and inducing the differentiation of desired cells from the three-germ-layer cells will be the most efficient differentiation method. In particular, in order to increase the utility of stem cells as a cell therapeutic agent, a technique of efficiently inducing the differentiation of stem cells into specific cells is required.
In recent years, a method of promoting the differentiation of human pluripotent stem cell-derived embryoid bodies by inhibiting their mTOR (mammalian target of rapamycin)(Zhou J et al., Proc Natl Acad Sci USA. 106(19):7840-5, 2009), and a method of promoting the differentiation of human pluripotent stem cells by inhibiting their telomerase (Yang C et al., Stem Cells. 26(4):850-63, 2008), have been reported. However, the method of inhibiting mTOR is a technique that does not act on a cell receptor, but acts on a signaling protein, and the method of inhibiting telomerase is also a technique that does not act on a cell receptor, but acts on the telomere-maintenance enzyme telomerase. In this method, non-specific differentiation into any other cell type, including endoderm, mesoderm and ectoderm, is promoted. In order to control the differentiation of human pluripotent stem cell-derived embryoid bodies, it is most important to develop a technique that acts on a cell receptor. This is because a technique that acts on a signaling protein or enzyme is highly likely influenced by other intracellular factors that influence signaling or enzymatic activity, compared to a technique that acts on a cell receptor, and because an operation for controlling the degree of differentiation can be relatively difficult. However, controlling the differentiation of human pluripotent stem cell-derived embryoid bodies by use of a cell receptor is not yet known.
Accordingly, the present inventors have made extensive efforts to induce the differentiation of stem cells into desired specific tissue or cells in order to increase the utility of stem cells as a cell therapeutic agent, and as a result, have found that the selective differentiation of human pluripotent stem cells into endoderm and mesoderm is promoted by inhibiting the expression of a CXCR2 receptor, thereby completing the present invention.